Endophyte communities vary in the needles of Norway spruce clones

f u n g a l b i o l o g y 1 1 7 ( 2 0 1 3 ) 1 8 2 e1 9 0

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Endophyte communities vary in the needles of Norway spruce clones Tiina RAJALA*, Sannakajsa M. VELMALA, Tero TUOMIVIRTA, Matti HAAPANEN, € Michael MULLER, Taina PENNANEN Finnish Forest Research Institute, Vantaa Research Unit, P.O. Box 18, FI-01301 Vantaa, Finland

article info

abstract

Article history:

Endophytic fungi show no symptoms of their presence but can influence the performance

Received 27 September 2012

and vitality of host trees. The potential use of endophytes to indicate vitality has been

Received in revised form

previously realized, but a standard protocol has yet to be developed due to an incomplete

26 November 2012

understanding of the factors that regulate endophyte communities. Using a culture-free

Accepted 11 January 2013

molecular approach, we examined the extent to which host genotype influences the

Available online 30 January 2013

abundance, species richness, and community composition of endophytic fungi in Norway

Corresponding Editor:

spruce needles. Briefly, total DNA was extracted from the surface-sterilized needles of 30

Paola Bonfante

clones grown in a nursery field and the copy number of the fungal internal transcribed spacer (ITS) region of ribosomal DNA was estimated by quantitative PCR. Fungal species

Keywords:

richness and community composition were determined by denaturing gradient gel electro-

Ectomycorrhizal fungi (ECM fungi)

phoresis and DNA sequencing. We found that community structure and ITS copy number

Fungal endophytes

varied among spruce clones, whereas species richness did not. Host traits interacting with

Host genotype

endophyte communities included needle surface area and the location of cuttings in the

Lophodermium piceae

experimental area. Although Lophodermium piceae is considered the dominant needle

Needles

endophyte of Norway spruce, we detected this species in only 33 % of samples. The

Norway spruce (Picea abies)

most frequently observed fungus (66 %) was the potentially pathogenic Phoma herbarum. Interestingly, ITS copy number of endophytic fungi correlated negatively with the richness of ectomycorrhizal fungi and thus potential interactions between fungal communities and their influence on the host tree are discussed. Our results suggest that in addition to environmental factors, endophyte communities of spruce needles are determined by host tree identity and needle surface area. ª 2013 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Introduction Endophytic fungi can infect live needles of coniferous trees without causing any visible signs or symptoms of disease (Petrini et al. 1993). By definition, endophytes inhabit the tissues of healthy plants for all or part of their life cycle (Wilson 1995). Several species have shown to be mutualistic and enhance resistance of the host plant against herbivores

and pathogens (Webber 1981; Carroll 1988; Clay 1988; Petrini et al. 1993; Redman et al. 2002; Arnold et al. 2003; Ganley et al. 2008; Torres et al. 2012), especially those infecting grasses (Saikkonen et al. 2010). Some endophytes are dormant litter € ller et al. 2001; Korkama-Rajala et al. 2008; decomposers (Mu Promputtha et al. 2010), whereas others are latent pathogens that activate during periods of host stress (Carroll 1988; Saikkonen et al. 1998). Endophytes may also affect their hosts

* Corresponding author. Tel.: þ358 29 534 5407. E-mail address: [email protected] (T. Rajala). 1878-6146/$ e see front matter ª 2013 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.funbio.2013.01.006

Endophyte communities vary in the needles of Norway spruce clones

through indirect effects on other host-associated symbionts. There is some indication that endophytes can reduce mycorrhizal colonization in grasses (Chu-Chou et al. 1992; Guo et al. 1992; Mack & Rudgers 2008) via chemical inhibition, altering plant nutritional requirements or redirection of plant resources away from mycorrhizal fungi. In woody plants, the relationship between the above ground endophytes and below ground mycorrhizal fungi is even less studied. Investigations of Norway spruce clones showed that the slowgrowing spruce clones had more saprophytic endophytes but fewer ectomycorrhizal (ECM) fungi than the fast-growing clones (Korkama et al. 2006; Korkama-Rajala et al. 2008). Different host species support different fungal endophyte communities (Hata & Futai 1996). Communities are also known to vary among individuals of the same host species, even when growing in the same site (Deckert & Peterson 2000). Interactions in grasses are highly specific since their endophytes are usually transmitted vertically via host seeds (Clay & Schardl 2002; Saikkonen et al. 2003, 2010), unlike those of woody plants that infect new hosts via spores (Petrini 1991). Studies of half-sib families of birch and Douglas fir have shown that the frequency of infection by a particular fungal endophyte or strain depends on the maternal genotype of the host (Todd 1988; Elamo et al. 1999; Ahlholm et al. 2002; Saikkonen et al. 2003). Likewise ECM colonization is a heritable trait in poplar (Tagu et al. 2001; Courty et al. 2011) and spruce (Velmala et al. 2013). Given that the intensity (i.e., quantity) and species composition (i.e., quality) of endophyte infections may affect host fitness, the role played by host tree genotype in determining endophyte community structure should be explored more fully. Although endophytes are a highly diverse group of fungi, only one or a few species dominate a given host tree (Sieber 2007). Lophodermium piceae is the most common endophyte in Norway spruce, where it typically infects more than 50 % of healthy needles (Barklund 1987; Sieber 1988; Livsey 1995; € ller et al. 2001). This species is not considered harmful to Mu its host (Barklund 1987) and is believed to enhance growth in Norway spruce (Korkama-Rajala et al. 2008). Most studies concerning endophyte communities are, however, based on pure cultures isolated on artificial media, and it is difficult to determine the extent to which their results are representative of natural infections in terms of species abundance and occurrence. This study is an attempt to bridge the gap from laboratory bench to tree stand and characterize endophyte communities more directly in relation to host tree genotype. We aimed to determine whether host tree genotype is significantly correlated with endophyte infections of needles from clonal material (i.e., cuttings) of Norway spruce. We were also interested in the endophytic species composition in young spruces growing in a tree nursery and their possible interaction with mutualistic ECM fungi, as this may partly determine vitality of seedlings in the future. We hypothesized that (i) communities of needle endophytic fungi vary among Norway spruce clones, (ii) abundance and species richness of spruce needle endophytes are heritable traits, and if associated with reduced host fitness, these traits can be used in tree breeding, (iii) L. piceae is the dominant fungal endophyte in fresh needles of young spruce cuttings, and (iv) spruces having substantial and diverse endophytic communities harbour

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poor ECM communities. Briefly, endophyte communities inhabiting the needles of 30 Norway spruce clones growing in a nursery field were determined in terms of species composition and relative abundance. We considered the fungal DNA extracted from fresh, healthy looking surface-sterilized needles to be an indication of endophytic infections and copy number of fungal rDNA internal transcribed spacer (ITS) as an approximate indicator of their abundance. Community structure was analysed via denaturing gradient gel electrophoresis (DGGE) and the main fungal species were identified by sequencing excised DGGE bands and comparing them to public databases.

Material and methods Plant material The study material consisted of rooted cuttings from 30 Norway spruce trees. The ortets were a sample of secondgeneration candidates selected for a Finnish breeding program. The clones were inoculated with a mix of ECM fungi (Laccaria sp., Piloderma sp., Amphinema byssoides, Paxillus involutus, and Cadophora finlandia) two growing seasons prior to the needle sampling and part of the cuttings were used earlier in a study of host tree genotype in relation to ECM fungi (Velmala et al. 2013). For the purpose of this experiment, clone cuttings were grown outdoors in a nursery field for two growing seasons in a randomized complete block design. The edge of a mature Norway spruce forest was approximately 200 m from the site.

Surface-sterilization Five cuttings from each clone were randomly selected and healthy looking needles from twigs of age class 2 (flushed in the spring of 2009) were collected in November 2010. Needles were initially sonicated for 1 min in 0.2 % Tween to dislodge dirt and epiphytes attached to needle surface. The wax layer was noted to be intact after the short sonication. After that, a commonly used surface-sterilization protocol (e.g., Sieber € ller et al. 2001) was followed and needles were shaken 1988; Mu for 1 min in 70 % ethanol, soaked for 4 min in 5 % sodium hypochlorite, shaken for 1 min in 70 % ethanol again, and then rinsed with 70 % ethanol. The brown base of each needle was excised after surface-sterilization. To check whether any remaining epiphytes could be amplified on the needle surface after the sterilization procedure, surface-sterilized needles were shaken for 10e30 min in a water/dilution buffer solution. Four microlitre of the solution was then used as template in PCR amplification using a highly efficient and robust DNA polymerase (Phire Plant Direct PCR Kit, Thermo Scientific) and the cycling conditions described in Velmala et al. (2013). No amplification was observed in the screening PCR reactions.

Determination of the needle biomass, length, and surface area Needle biomass was determined as the fresh weight of 25 needles per cutting and needle length by measuring five needles per cutting to the nearest millimetre (Table 1).

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T. Rajala et al.

Table 1 e Characteristics of the 2-y old needles in Norway spruce clone cuttings. Mean Needle biomass (mg/25 needles) Needle length (mm) Needle surface area (mm2)

Median

42.4

40

8.44 15.8

8.55 15.2

MineMax

P-valuea

10e140

0.018

4.4e17.4 4.5e52.5

0.331 0.331

a One-way ANOVA: difference between clones.

In order to calculate needle surface area from needle length, a subset of ca. 1000 needles was used to determine the relationship of needle length and area. Needle surface area was estimated from needle projections by multiplying each needle projection area by the coefficient 2.74 (Riederer et al. 1988). Projections were obtained by scanning (Epson Perfection V700 Photo, Seiko Epson Corporation, Nagano, Japan) detached needles of age class II (year 2009) and measuring areas with the image analysis software ASSESS (American Phytopathological Society, Minnesota, USA). The relation between needle length and surface area was linear y ¼ 3.805x  8.504, adjusted R2 0.797 (where x is needle length and y surface area).

DNA extractions Total genomic DNA was isolated from fresh needles homogenized with a mortar and pestle in liquid nitrogen (80 mg) using the NucleoSpin Plant II (MachereyeNagel) DNA extraction kit. Of the two optional lysis buffers, we used PL1. The standard protocol given by the manufacturer was slightly modified in that incubation time was increased to 30 min and the DNA elution step was repeated once with 100 ml PE buffer. DNA samples were precipitated by adding 0.6 vol of a PEGeNaCl solution [20 % PEG (w/v), 2.4 M NaCl] and incubated on ice for 20 min. Finally, samples were centrifuged at 16 000g for 20 min and the pellets were washed with 70 % ethanol, dried, and resuspended in 1:10 TE buffer (0.6 mM Tris/HCl, 0.1 mM EDTA) prior to storage at 20  C.

Quantitative PCR The 20 ml qPCR reaction contained 10 ml Maxima SYBR Green qPCR Master Mix (Fermentas), 0.375 mM of ITS1F (Gardes & Bruns 1993) and ITS2 (White et al. 1990) primers, and 1 ml of DNA template. The reaction was carried out on a Rotor-gene 6000 (Corbett Research) apparatus using a temperature program starting at 95  C for 10 min, followed by 40 cycles of 95  C for 15 s, 55  C for 30 s, and 72  C for 50 s, and a melting curve analysis. A dilution series with known amounts of Lophodermium piceae was used for a standard curve (CT ¼ 3.35  log(conc)  1.03, r2 ¼ 0.994, reaction efficiency 0.989). All reactions were repeated four times, including negative controls.

DGGE analysis and sequencing Technical replicates of qPCR products were combined prior to DGGE analysis. To make the amplicons suitable for DGGE, PCR

amplification was performed with a modified ITS1F primer including a 40 bp GC-clamp (Muyzer et al. 1993) and an unmodified ITS2 primer. The 20 ml PCR reactions contained 2 ml 10 DreamTaq buffer, 0.2 mM of each dNTP, 0.5 mM of primers, 1 u DreamTaq, (Fermentas) and 1 ml of qPCR products. Cycling parameters consisted of a 3 min initial denaturation at 95  C followed by 10 cycles of 95  C for 30 s, 58  C for 30 s, 72  C for 50 s, and a final extension of 72  C for 5 min. PCR products were analysed in DGGE using the INGENY phorU system (Ingeny). The denaturing gradient of 7.5 % (w/ v) acrylamide gel was 18e58 % produced with 100 % solution containing 40 % deionized formamide (SigmaeAldrich) and 7 M urea (BioRad). The electrophoresis was performed in TAE buffer (40 mM Tris-acetate, pH 8, and 1 mM EDTA) for 18 h at 75 V and 60  C. The gels were stained with SYBR Gold (Molecular Probes) and visualized with blue light on a SafeImager transilluminator (Invitrogen). DGGE bands of interest were excised and eluted into 100 ml of sterile water. The amplicons were reamplified with ITS1FGC/ITS2 primer pair and rerun in DGGE as described above. This procedure was repeated up to five times until only the excised bands were visible in sample profiles. They were then amplified with primers ITS1/ITS2, purified (HighPure PCR Product Purification kit, Roche), and sequenced using PCR primers by the Macrogen Sequencing Service. Contigs representing consensus DNA sequences were assembled and edited in Geneious Pro 4.8 (Biomatters, available from http:// www.geneious.com/) and blasted against fungal sequences in GenBank. Sequences are deposited in GenBank under the accession numbers JX236473eJX236502.

Data analysis Images of DGGE profiles were analysed with GelCompar II (version 5.1, Applied Maths). The presence or absence of bands in each sample profile was determined with a band matching optimization of 0 % and band position tolerance of 1 %. The binary data of band classes were then visualized via nonmetric multidimensional scaling (NMDS). Examination of the endophyte community structure was based on a three-dimensional NMDS approach using the default BrayeCurtis (Bray & Curtis 1957) dissimilarity index as based on the rankindex comparison, it had the highest rankorder similarity for our community data with the environmental variables provided. One-dimensional NMDS was used to condense multispecies abundances for each spruce clone into a single sequence of scores used to estimate heritability. Additionally, a permutational multivariate analysis of variance (MANOVA) of distance matrices was conducted with the function adonis to test for the effect of clone, location of cutting in the nursery field, needle biomass, and needle area on the multivariate endophyte community. Distance matrices analysed in MANOVA were based on BrayeCurtis dissimilarity indices and significance of the test was determined according to sequential sums of squares from 4999 permutations of the raw data. To analyse host genotype effects on needle characteristics (i.e., biomass, length, and area), we fitted a one-way analysis of variance (ANOVA) with the function aov and clone as the only explanatory variable. We fitted a linear model lm to determine whether clone, needle surface area or location accounted

Results

ITS copies l-1

10000 10 8 6 4 2

Nr of band classes

B

12

0

5000

A

330 387 215 301 30559 30586 30648 30739 30784 30860 64 219 298 431 452 30560 30623 30715 30857 20 46 261 435 30608 30652 30813 53 88 428 30631

0

for differences in ITS copy numbers. Due to the strong colinearity between needle biomass, length, and surface area, only needle surface area was used in the models. We considered surface area the most interesting needle-related variable because it is the interface for endophytic infections mediated via spores. Location was used as a spatial grouping factor explaining variation due to placement in the nursery field, accounting for potential microenvironmental variation and exposure to fungal spores from the nearby forest. To maintain normally distributed residuals, we log transformed the number of ITS copies. To study clonal and spatial effects in discrete data such as richness (i.e., DGGE band class counts) we used a generalized linear model glm assuming a Poisson error distribution and log-link function. The model included clone, needle surface area, and location as explanatory variables. No interaction terms were included in the models as they were clearly nonsignificant. Linear relationships between traits were analysed using Pearson’s correlation statistics and as above, the number of ITS copies was log transformed. The relationships between the present endophyte abundance and richness data and the former ECM fungal colonization and richness data (Velmala et al. 2013) were tested with a dataset comprising the mean values for each of 30 clones, since endophytes and ECM fungi were not analysed from exactly the same cuttings. All statistics and ordinations described above were performed with the R 2.15.0 software (R Development Core Team 2012). Functions rankindex, adonis, and metaMDS are found in the ‘vegan’ library (Oksanen et al. 2012). Estimates of heritability are used in tree breeding programs to predict the genetic gain from clonal selection experiments (White et al. 2007). Broad-sense heritability is the fraction of selection differential that will be realized as genetic gain when the selected individuals are deployed as asexually propagated plants. Broad-sense heritability was estimated as H2 ¼ s2Clone/(s2Clone þ s2Block þ s2W residual error) and standard errors were determined using an approximation procedure (Dickerson 1969). Variance components were estimated by the SAS MIXED (9.2) procedure using the REML algorithm (Littell et al. 1996).

185

15000

Endophyte communities vary in the needles of Norway spruce clones

Spruce clone

Fig 1 e (A) ITS copy number and (B) richness of endophytic fungi in DNA samples extracted from needles (80 mg f.w.) of Norway spruce clones. Results are presented as Box and Whisker plots which show medians (bold line), lower and upper quartiles (box bottom and top), largest and smallest values (whiskers), and outliers (circles).

No statistically significant differences were observed with respect to species richness among clones (Fig 1B, Table 3). Needle surface area was significant in the model explaining species richness (Table 3), and negative correlation between surface area and richness was almost significant (r ¼ 0.153, t ¼ 1.88, df ¼ 148, P ¼ 0.062). No correlation was found between endophyte richness and needle biomass (r ¼ 0.052, t ¼ 0.488, df ¼ 148, P ¼ 0.525), neither with endophyte richness and number of needles in samples (r ¼ 0.061, t ¼ 0.740, df ¼ 148, P ¼ 0.462). Broad-sense heritability values of endophytic ITS copy number and species richness were very low, 3.5 % (6.5 %) and 0.9 % (6 %), respectively. This indicates that almost none of the variation in these measured characteristics is genetically determined.

ITS copy number and species richness Fungal ITS copy number varied among spruce clones (Fig 1A). In addition to spruce clone, needle surface area was a significant factor explaining variation in ITS copy number, whereas nursery container (location) was not (Table 2). ITS copy number in an 80 mg sample correlated negatively with the average needle surface area (r ¼ 0.333, t ¼ 4.30, df ¼ 148, P < 0.001) and needle biomass (r ¼ 0.365, t ¼ 4.78, df ¼ 148, P < 0.001). Number of needles in the sample correlated positively with the copy number (r ¼ 0.415, t ¼ 5.55, df ¼ 148, P < 0.001). Therefore, when expressed as total needle surface area in the sample, the correlation between surface area and ITS copy number was positive (r ¼ 0.240, t ¼ 3.00, df ¼ 148, P ¼ 0.003). In general, higher copy numbers were found in species poor samples, i.e., a negative correlation between copy number and species richness (r ¼ 0.213, t ¼ 2.65, df ¼ 148, P ¼ 0.009).

Community structure of endophytic fungi According to a permutational MANOVA, clone explained 24 % of the observed variance in community structure (Table 4),

Table 2 e ANOVA: effect of explanatory variables on ITS copy number of needle endophytic fungi according to a linear model fit.

Clone Needle surface area Location Residuals Adjusted R2

Df

SS

MS

F-value

P-value

29 1 24 95 0.27

24.2 5.13 10.1 33.9

0.833 5.13 0.420 0.357

2.33 14.3 1.18

0.001 <0.001 0.283

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T. Rajala et al.

149 120 119

25.2 4.74

76

P-value

Dot(60)

130 104 99.7

0.668 0.029

24 0.38

19.4

95

80.3

Needle surface 38 area Needle biomass unc(47) 74

Sis(68)

0.5

Resid. dev

Lop(64)

46

Lir(72)

Rho(44)

80 67

53

0.0

29 1

Resid. df

Wal(39)

a

45 Par(52)

Ram(69)

0.733

48

Alt(59)Rho(51)

Cha(54)

unc(77)

Pho(71)

Ram(62)

83

ITS copy numbers

−0.5

a Chi-squared test. b (Null deviance  residual deviance)/null deviance.

Cap(57)

Lac(49)

Cad(56)

and the three-dimensional NMDS plot illustrates separation of the endophyte communities among the spruce clones (Fig 2). However, heritability of the community structure among different clones was zero according to one-dimensional NMDS scores (data not shown). It should be noted that the data exposed to the MANOVA were presence/absence of DGGE band classes whereas heritability was calculated for onedimensional NMDS score values. Other variables interacting significantly with fungal community structure included location (R2 ¼ 0.230) and needle surface area (R2 ¼ 0.012) (Table 4). Needle biomass differed significantly among clones, whereas length and surface area did not (Table 1). Forty-one DGGE bands were excised and sequenced for identification purposes. The most frequently found endophyte was Phoma herbarum, which represented 14.9 % of all DGGE bands, was found in 66 % of samples (Table 5). Alternaria alternata and members of Capnodiales constituted 11 % and 7.8 % of the bands, respectively. Around 7.5 % of the bands were identified as Lophodermium piceae, found in 33 % of samples. Fungal species typical for small needles were similar to sequences of Cadophora luteo-olivaceae and members of Capnodiales (Fig 2). Sistoderma brinkmannii and Cryptococcus friedmannii were associated with high species richness (Fig 2), but no single species explained the high ITS copy numbers found in some clones (data not shown).

−1.0

NULL Clone Needle surface area Location Pseudo R2b

Deviance

Fungal richness Cry(41)

NMDS3

Df

1.0

Table 3 e Analysis of deviance: effect of explanatory variables on richness of needle endophytic fungi according to a generalized linear model fit.

−1.5

−1.0

−0.5

0.0

0.5

1.0

NMDS1

Fig 2 e Three-dimensional NMDS of needle endophytic fungal communities among Norway spruce clones. Ellipses are drawn based on standard errors of mean scores of clones. Vectors indicate the significant variables (P < 0.1) and their increasing gradient in the ordination space. Loadings of each fungal species (or DGGE band class if species identity was not determined) are presented. See Table 5 for complete species names.

correlation was found between endophyte ITS copy number and ECM richness (r ¼ 0.328, t ¼ 1.84, df ¼ 28, P ¼ 0.077). No significant linear relationships were found between copy number and ECM colonization level (r ¼ 0.172, t ¼ 0.926, df ¼ 28, P ¼ 0.363), endophyte richness and ECM colonization level (r ¼ 0.172, t ¼ 0.926, df ¼ 28, P ¼ 0.363) or species richness of endophytic and ECM fungi (r ¼ 0.157, t ¼ 0.841, df ¼ 28, P ¼ 0.408).

Discussion Interaction between endophytic fungi and ECM fungi Endophytes in needles of different spruce clones Endophyte fungal data from this experiment were combined with those of Velmala et al. (2013), who investigated ECM communities on the same spruce clones. A weak negative

Table 4 e Significance and explanatory power of explanatory variables on the variation in endophyte community structure according to permutational MANOVA. Variable

Df

SS

Clone 29 9.89 Needle surface area 1 0.510 Location 24 9.47 ITS copies 1 0.131 Residuals 94 21.2 Total 149 41.2

MS

F-value

R2

P-value

0.341 0.510 0.395 0.131 0.225

1.51 2.26 1.74 0.580

0.240 0.012 0.230 0.003 0.515 1.00

<0.001 0.034 <0.001 0.767

This study showed that communities of endophytic fungi can vary among young Norway spruces. Species were identified via culture-free DNA methods, which enabled a more comprehensive view of the fungal endophytes occurring in fresh needles. The number of endophytic fungal taxa did not differ significantly among clones, whereas ITS copy numbers determined by qPCR and the community structure did. Traditionally, fungal abundance has been estimated by measuring hyphal mycelia or the colonization of host tissue via microscopy. Measuring fungal abundance by microscopy is, however, difficult and prone to subjective interpretation. Similarly, techniques based on the measurement of chitin, ergosterol or fungal phospholipid fatty acids are not ideal indicators of fungal biomass in field samples due to their nonspecificity (Sharma et al. 1977; Olsrud et al. 2007) and because the concentrations at which these compounds exist in

Endophyte communities vary in the needles of Norway spruce clones

Table 5 e Identity of DGGE band classes (according to closest GenBank match) representing fungal taxa found on surface-sterilized needles of Norway spruce, their proportion of all bands in DGGE gels (% of bands), and proportion of samples containing the band class (% of samples). DGGE band class 71 59 57 48 64 60 69 74 56 53 62 67 46 47 51 54 68 52 49 44 72 45 76 39 80 38 77 83 41 22 27 31 33

Identitya

% of bands

% of samples

Phoma herbarum Alternaria alternata Capnodiales nd Lophodermium piceae Dothioraceae sp. Ramularia sp. nd Cadophora luteo-olivaceae nd Ramularia sp. nd nd Uncultured fungus Rhodotorula sp. Chalara longipes Sistotrema brinkmannii Paraphoma sp. Laccaria laccata Rhodotorula sp. Lirula macrospora nd nd Wallenia sp. nd nd Uncultured fungus nd Cryptococcus friedmannii nd nd nd nd

14.9 11.0 7.8 7.5 7.5 5.3 5.0 3.9 3.8 3.5 3.2 3.0 2.9 2.9 2.9 2.0 2.0 1.8 1.7 1.4 1.4 0.9 0.9 0.6 0.6 0.5 0.5 0.5 0.3 0.2 0.2 0.2 0.2

66.0 48.7 34.7 33.3 33.3 23.3 22.0 17.3 16.7 15.3 14.0 13.3 12.7 12.7 12.7 8.7 8.7 8.0 7.3 6.0 6.0 4.0 4.0 2.7 2.7 2.0 2.0 2.0 1.3 0.7 0.7 0.7 0.7

nd, not determined by excising and sequencing DGGE bands. a Based on 97 % similarity to a sequence in GenBank.

the mycelia of various species in situ are unknown. Compared to traditional methods, qPCR offers a fast and sensitive way to detect and quantify fungi in environmental samples (Smith & Osborn 2009). Fungal DNA can be detected from small samples and analyses can be restricted to particular taxa by using specific PCR primers. The ribosomal ITS region is relatively easy to detect in small samples because it is abundant in fungal cells and several studies have used it as a target for quantification of biomass in environmental samples (Raidl et al. 2005; Manter & Vivanco 2007; Boyle et al. 2008; Tellenbach et al. 2010). Although the use of qPCR for analysing fungal biomass has been criticized because the ITS copy number may vary among fungal taxa (Hibbett 1992; Landeweert et al. 2003; Corradi et al. 2007), we did not find evidence of systematic bias and, furthermore, clones with high ITS copy numbers did not support particularly divergent communities according to NMDS. Therefore, we believe that variation in ITS copy number among spruce clones in this study indicates real differences in relative abundance of endophytic fungi.

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Our result that needles with high fungal abundance had lower species richness may reflect competitive interactions among endophytes. According to the classical ecological theory ‘Competitive Exclusion Principle’, endophytes compete for the same realized niche and this competition can be played via chemical antagonism. For example, Lophodermium piceae forms black zone lines in cultures and needles as a result of chemical reactions against other strains or species (Stephan & Osorio 1995). Fungal competition can also be mediated by induced host defence e a mechanism that likely plays a role in the interactions of plant-associated microbes (Saunders &  n et al. 2011). Kohn 2009; de Roma Endophyte community structures varied among spruce clones according to DGGE profiling. These results are in line with those concerning Douglas fir or birch genotypes and a particular endophyte genotype (Todd 1988; Elamo et al. 1999; Ahlholm et al. 2002; Saikkonen et al. 2003), implying that host susceptibility is genetically determined. The influence of host genotype on endophytes was lower than the effect on root ECM fungi (Velmala et al. 2013), probably because of a less specific interaction between endophyte and host, and a stronger influence of environmental factors on airborne dispersal of endophyte spores compared to the growth of ECM mycelia. Needle endophytes typically occupy one or a few cells (Carroll 1986; Suske & Acker 1987, 1989) whereas ECM fungi colonize whole root tips and probably protect the root from a diverse array of opportunistic and short-lived colonizers. Therefore, it may well be that the species detected in mycorrhizal root tips better reflect the fungi living in the target tissue than do those detected in needles. Although clone explained a quarter of the observed variation in endophyte community structure, there are other influential factors to consider. For example, the location of a cutting in the experimental field also accounted for a significant portion of variation in species richness, and it is known that exposure to inocula and microclimate influence endophyte distribution and infection rates (Carroll & Carroll 1978; Deckert & Peterson 2000). Another important factor influencing endophyte community structure was needle size. Fungal abundance was highest in samples where the average needle biomass was small. Accordingly, those samples contained more needles and consequently a larger surface area. If endophyte infections are restricted to a few epidermal and nearsurface mesophyll cells, e.g., L. piceae (Carroll 1986; Suske & Acker 1987, 1989), then it is natural that the number of infections correlates with needle surface area. It should be noted that average needle length and surface area did not differ statistically among clones in our study whereas needle biomass did. Although needle diameter and width depend on light conditions (Stenberg et al. 1999) and can vary in different parts of a tree (Sellin 2000), needle length of Norway spruce does not respond strongly to light availability (Niinemets & Kull 1995; de Chantal et al. 2003; Metslaid et al. 2007). Thus, it seems that environmental impacts on endophyte communities are mediated mainly via quality and quantity of the sporal load and host genotype effects are rather related to needle biomass. Variation in defence compounds and structures is known to affect endophyte colonization and assembly (Bailey et al. 2005; Saunders & Kohn 2009) but these were not measured in our study.

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Endophyte species detected in spruce needles Endophyte communities found here differed from those commonly found by culturing surface-sterilized needles of Norway € ller et al. spruce (Barklund 1987; Sieber 1988; Livsey 1995; Mu 2001; Korkama-Rajala et al. 2008). Lophodermium piceae was present but was not the dominant needle endophyte, as is generally reported. We suggest that the low occurrence of L. piceae is due to unfavourable climatic conditions in the sunny open nursery field or limited dispersal of spores from the nearby spruce forest. Given that the most frequently observed fungal species (Phoma herbarum and Alternaria alternata) are typically isolated from Pinus and Quercus seedlings in forest nurseries (MartinPinto et al. 2004), relative abundance of endophytic species in spruce needles may change after out-planting. On the other hand, different identification techniques may partly explain the discrepancy between previous studies and the results presented here. Arnold et al. (2007) noted that several commonly cultured taxa were not frequently detected in DNA extractions from surface-sterilized needles of loblolly pine. They also found that a culture-free approach revealed higher fungal diversity compared to culture methods, as did Korkama-Rajala et al. (2008). Observations can be explained by the fact that direct DNA extraction may reveal sequences of dead fungi, which are able to colonize internal tissues of needles but tend to die soon and therefore remain undetected by culture methods. Furthermore, PCR may amplify some species more than others, whereas culturing can bias estimates of community composition because fungi may differ in their ability to grow in vitro. Sieber (2007) suggested that while extraction of endophytic fungal DNA directly from surface-sterilized needles provides an effective characterization of endophyte diversity, any remnant epiphytic DNA may skew results. The screening PCR of surface-sterilized needles failed to amplify any fungal DNA and it is highly likely that needle surfaces did not contain amplifiable epiphytic DNA sequences after the surfacesterilization procedure. One of the species found in DNA extracted from surface-sterilized needles was similar to Laccaria laccata, a common ECM species in forest tree nurseries in Finland (Flykt et al. 2008). Laccaria laccata was also the most common ECM species in our study (Velmala et al. 2013) and its fruiting bodies were frequently observed growing in the nursery containers, meaning that spores of L. laccata were abundant in the air during the experiment. It is possible that the sterilization procedure failed to remove fungal spores of L. laccata and other species sheltering in stomata, although results suggest that the proportion of species from stomata remained small, as L. laccata represented only 1.7 % of observations. Nevertheless, some of the species found in this investigation may represent abundant spore producers that do not infect needles. Other ITS sequences frequently detected but unnamed to the species level (e.g., the Capnodiales- and Dothioraceae-like sequences) may represent previously unknown endophytes that have been overlooked by earlier investigations based on isolation and culturing methodology.

Interaction between endophytes, ECM fungi, and host vitality When endophyte abundance and species richness were compared to the colonization level and richness of ECM fungi of

T. Rajala et al.

the same clonal origins (Velmala et al. 2013), a slightly negative correlation was found between endophyte abundance and ECM richness. Relationship between endophyte abundance and ECM colonization was not significant, probably because almost all short roots were mycorrhizal. Interestingly, our previous studies (Korkama et al. 2006; Korkama-Rajala et al. 2008) showed that fast-growing Norway spruce clones harboured in average 22 % more ECM fungi but 46 % less saprotrophic needle endophytes than slow-growing clones in the field. Accordingly, above ground fungal endophytes associated with grasses are known to inhibit below ground arbuscular mycorrhizal colonization (Chu-Chou et al. 1992; Guo et al. 1992; Mack & Rudgers 2008), although the response depends on the accessible recourses and identity of arbuscular fungi (Larimer et al. 2012). Several mechanisms may explain negative effects of endophytes on mycorrhizal fungi. As discussed by Mack & Rudgers (2008), endophytes may trigger production of alkaloids and plant defence chemicals, which inhibit the growth of mycorrhizal fungi. Moreover, endophytes may change resource allocation between endophytic and mycorrhizal fungi. Thus, our finding highlights the question concerning the role of needle endophytes in interactions between host tree and microbes and also in tree growth. Lophodermium piceae is not considered harmful to its spruce host (Barklund 1987), whereas Phoma herbarum, the dominant endophyte in our study, may be pathogenic (Lilja et al. 2005). A negative correlation between needle endophyte colonization and Norway spruce vitality has also been observed by Barklund & Rowe (1983) and Sieber (2007) who observed that the onset of needle senescence begins as soon as endophyte density exceeds a certain threshold value. As such, high needle endophyte abundance may be an indicator of diminished resistance and vitality in spruce.

Conclusions Our findings support the hypothesis that endophytic fungal communities vary among spruce clones and suggest some relationship between host trees and their endophyte communities. The observed variation in endophyte composition and abundance in the needles of young spruce cuttings is partly explained by host tree identity, needle length, and surface area. However, none of the endophyte characteristics were significantly heritable in the studied spruce population and environmental factors, such as exposure to spores and microclimate, seem to largely shape the endophytic community of spruce needles. Lophodermium piceae commonly isolated from Norway spruce needles was detected in only one-third of samples while the dominant species was the potentially pathogenic Phoma herbarum. The implied lower occurrence of L. piceae may be related to unfavourable environmental conditions in the nursery field, limited spore dispersal from the nearby spruce forest, or potential biases due to identification approaches used in this (i.e., culture-free DNA based) and earlier studies (i.e., in-vitro culture). Interestingly, the relationship between endophyte abundance and ECM species richness was negative, suggesting indirect antagonistic interactions between endophytic and ECM fungi. Future research should evaluate the effects endophytes may have on their spruce hosts, and whether the high

Endophyte communities vary in the needles of Norway spruce clones

abundance of certain needle endophytes can reduce spruce vitality.

Acknowledgements The study was funded by The Academy of Finland (Project € yrynen and other Number 128229). We are grateful to M. Ha laboratory technicians of the Finnish Forest Research Institute for help with the sample preparation and analysis, Prof. em. T. Kurkela for proving use of ASSESS software, Dr M. Hardman for revising the text and two anonymous referees for useful comments.

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